捕食线虫真菌少孢节丛孢中milRNA的发现及功能机制研究

发布时间：2018-02-24 10:05

本文关键词： 少孢节丛孢 milRNA milR289 QDE-2 捕器 HITS-CLIP RIP　出处：《云南大学》2016年博士论文　论文类型：学位论文

【摘要】：非编码RNA广泛存在于原核生物和各种真核生物中,在生物体的生长发育衰老疾病等生物学机制中都发挥重要的调控作用。MiRNA是非编码RNA中研究得最为清楚的一类small RN A,大小在21 nt左右,在动植物中的功能和调控机制都有比较系统的报道,并已应用到各物种的生物学功能机制研究和人类疾病治疗等方面。目前真菌中已经报道了microRNA-like RNA,但其生物学功能还不清楚。本文在捕食线虫真菌少孢节丛孢(简称少孢)中发现milRNA,并且证明milR289能够以qde-2等基因为靶基因。Qde-2等基因在少孢生活史转化中起到重要作用,即在少孢形成捕器三维菌网的过程中发挥作用。我们通过small RNA组测序和northernblot证明少孢中存在small RNA,根据其序列信息和northern blot杂交结果,发现这些small RNA与miRNA结构类似,将其命名为microRNA-like RNA (milRNA)。RIP实验表明milR289绑定在Argonaute蛋白QDE-2上发挥作用。QDE-2是形成RISC复合体的核心成分,在RNAi的过程中起到剪切或抑制靶基因的功能。我们通过生物信息学预测、HITS-CLIP和RIP-RT-PCR发现了nilR289的三个可能的靶基因,有意思的是,qde-2 mRNA自身就绑定在RISC复合体成分QDE-2上。进一步通过烟草瞬时表达系统体外验证了QDE-2是mi1R289的靶基因。并且QDE-2的敲除实验发现其在urea和线虫提取液、活线虫诱导的捕器形成过程中都具有重要作用。综上,我们首次在捕食线虫真菌中发现milRNA,首次在丝状真菌领域证明milRNA的靶基因并阐述其功能及机制,并且该机制在捕器形成过程中发挥重要作用。功能机制研究发现,milR289能够以RISC核心蛋白QDE-2为靶基因,从而在RNAi过程中形成一个,milRNA与QDE-2 (RISC)作用调控的环路,即milR289绑定QDE-2形成RISC又作用于qde-2基因来抑制RNAi作用。这条功能机制在植物中有过类似报道,表明真菌中milRNA和植物中miRNA调控机制上可能存在保守性。
[Abstract]:Non-coding RNA exists widely in prokaryotes and various eukaryotes. It plays an important regulatory role in biological mechanisms, such as growth, development, senescence and other biological mechanisms. MiRNAs are the most clearly studied class of small RNA in non-coding RNA, the size of which is about 21 NT. The functions and regulatory mechanisms in plants and animals have been systematically reported. MicroRNA-like RNAs have been reported in fungi, but their biological functions are not clear. In this paper, the predatory nematode fungus oligosporium (abbreviated as oligosporium) has been used to study the biological function of RNAs. MilRNAs were found in oligospora, and it was proved that milR289 can play an important role in the transformation of oligosporal life cycle by using qde-2 isobars as the target gene. Qde-2 and other genes play an important role in the transformation of oligosporal life cycle. We proved that small RNAs existed in oligospora by small RNA sequencing and northernblot. According to the sequence information and northernblot hybridization results, we found that these small RNA were similar to miRNA structure. It was named microRNA-like RNA millet RNA.RIP experiment showed that milR289 binding on Argonaute protein QDE-2. QDE-2 is the core component of RISC complex. We found three possible target genes of nilR289 by bioinformatics prediction, HITS-CLIP and RIP-RT-PCR. It is interesting to note that the Qde-2 mRNA itself is bound to the RISC complex component QDE-2. Further, the transient expression system of tobacco proved that QDE-2 is the target gene of mi1R289 in vitro, and QDE-2 knockout test found that it was found in the urea and nematode extracts. All of them play an important role in the process of trap formation induced by living nematodes. In summary, milRNAs were first found in nematode predatory fungi, and the target genes of milRNA were demonstrated in the field of filamentous fungi for the first time, and its function and mechanism were explained. This mechanism plays an important role in the process of trap formation. It was found that Mil R289 was able to target the RISC core protein QDE-2 in the process of RNAi, thus forming a loop regulating the interaction between QDE-2 and RISC during the process of RNAi. That is, milR289 binds QDE-2 to form RISC and acts on qde-2 gene to inhibit RNAi. This mechanism has been reported in plants, suggesting that milRNA and miRNA in plants may be conserved.
【学位授予单位】：云南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q933

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